Method for obtaining antigen-specific tr1 regulatory lymphocytes

ABSTRACT

The invention relates to a method for preparing antigen-specific Tr1 regulatory lymphocytes. The inventive method involves the use of artificial antigen-presenting cells, expressing a molecule from the HLA class II system and a human LFA-3 molecule and expressing none of the B7-1, B7-2, B7-H1, CD40, CD23 or ICAM-1 costimulation molecules.

[0001] The invention relates to obtaining regulatory CD4⁺ T lymphocyteswhich can be used for the prevention and the treatment of autoimmunediseases and inflammatory diseases.

[0002] Autoimmune diseases are the result of a deregulation of theimmune system, which manifests itself by an undesirable immune responseof an organism to its own antigens.

[0003] It has been attempted to manipulate the antigens which bringabout these pathologies or the aggressive T-cells which are specific forthese antigens, but the results obtained have frequently been verylimited owing in particular to a lack of knowledge of all the antigensimplicated in the pathology in question. This is because the actualauto-antigens, or the antigens which are responsible for inflammatorydiseases and autoimmune diseases, are not always known or differ betweenindividuals. The treatments which are currently used against thesediseases are either palliative treatments (insulin in the case ofdiabetes, antihistamine in the case of allergic disorders) or systemictreatments with anti-inflammatories (AINS) and/or immunosuppressants(glucocorticoids, cyclosporin, antibodies and the like). There istherefore clearly a need for an immunosuppressant treatment which ispotent, but limited to the affected organ or more precisely to thehyperactivity zone of the immune system.

[0004] Among the large number of agents implicated in the regulation ofimmune response, there are the CD4⁺ T lymphocytes, also referred to asT-helper lymphocytes. Traditionally, two main types of T-helperlymphocytes are distinguished: the Th1 lymphocytes, which are implicatedin the development of the cellular immune response, producepro-inflammatory cytokines such as interleukin-2 (IL-2) and γ interferon(IFNγ) and have macrophage-activating activity; the Th2 lymphocytes,which produce cytokines such as the interleukins IL-4, IL-6, IL-10 andIL-13, promote the secretion of antibodies.

[0005] Recent research, in which one of the inventors has participated,has managed to discover a novel category of regulatory T cells whoseproliferation is induced by the activation of CD4⁺ T cells in thepresence of interleukin 10 (IL-10) and which have been called Tr1lymphocytes (PCT application WO 97/42324). By continuing this research,it has been possible to isolate and characterize a novel subpopulationof regulatory T cells, which were called Tr1 lymphocytes (Groux et al.,Nature, 389, 737-742, 1997). This lymphocyte subpopulation has beenobtained by repeatedly activating CD4⁺ T cells in the presence of anantigen and of interleukin 10 (IL-10). When the Tr1 cells arerestimulated by the antigen used for their induction, they onlyproliferate weakly, produce very high amounts of IL-10, high amounts ofTGF-β (tumor growth factor β), very small amounts of IL-2 and no IL-4.When activated Tr1 cells are grown in the presence of other CD4⁺ Tcells, they suppress the proliferation of the latter in response to anantigen; this effect is due to the secretion of cytokines, in particularIL-10, by the Tr lymphocytes and not due to a direct action of thelatter on the CD4⁺ T cells; it can therefore be obtained without havingto know the antigen which is responsible for the proliferation of thesecells. This is of great advantage in the case of autoimmune diseases,where the treatment can thus be envisaged without it being necessary toknow the exact antigen against which the pathogenic cells are directed.

[0006] It has thus been observed in an experimental model of Crohn'sdisease in the mouse, in which the proinflammatory cells are directedagainst commensal bacteria of the gut flora, that the administration tothe animals of Tr1 cells which are directed against ovalbumin inconjunction with the administration of ovalbumin in the food allows theinstitution of chronic inflammation of the colon to be prevented.

[0007] Secondly, the inventors have demonstrated in recent research ondifferent animal models of Crohn's disease, of multiple sclerosis or ofgraft-versus-host reactions, that the inhibitory Tr1 cells were not onlycapable of preventing, but also of curing, these different pathologies.Moreover, they have observed that the Tr1 cells migrated specificallyand intensively into the different sites of inflammation. Thischaracteristic of the Tr1 cells represents an additional advantage inthe use of these cells as vectors for anti-inflammatory molecules in thesites of severe inflammation. Tr1 cells obtained from T cells of apatient are therefore potentially useful in the context of a celltherapy for regulating the immune response in this patient. They canthus be used in particular for preventing or treating not only theabovementioned autoimmune diseases and inflammatory diseases, butlikewise any other pathology which is characterized by an aberrantinflammatory response such as diabetes, psoriasis, atherosclerosis,rheumatoid polyarthritis or asthma; they can likewise be used in thetreatment of graft rejections or of a graft-versus-host reaction.

[0008] The method for obtaining Tr1 lymphocytes which is described inthe publication of Groux et al. and which is cited hereinabove requiresthe repeated stimulation of T cells with the antigen in the presence ofIL-10. This method is laborious and does not allow to obtain rapidlyfrom a patient a Tr1 lymphocyte population which can be used for atherapeutic treatment.

[0009] Different teams have reported the implication of differentcostimulation molecules in obtaining cells which have characteristicsresembling those of Tr1 cells, in particular in the production of IL-10;these stimulation molecules which may be implicated are very diverse andthe effects reported sometimes appear to be contradictory.

[0010] Thus, Bleijs et al. (Eur. J. Immunol., 29, 2248, 1999) haveobserved that costimulation by the interaction of LFA-1 with ICAM-1,ICAM-2 or ICAM-3 induces a high production of GM-CSF(granulocyte-macrophage colony-stimulating factor), of IFN-γ and ofIL-10 by T lymphocytes which are stimulated with an anti-CD3 antibody.The production of IL-10 is particularly high when the costimulation iscarried out by LFA-1/ICAM-1 interaction. Bullens et al. (Int. Immunol.13, 181-191, 2001) report that costimulation by CD58 induces theproduction of IL-10 and IFN-γ by T cells which are stimulated by ananti-CD3 antibody. Dong et al. (Nat. Med., 5, 1365, 1999) have describeda costimulation molecule which belongs to the B-7 family and is referredto as B7-H1, which induces preferentially the secretion of IL-10.

[0011] Van Gool et al. (Eur. J. Immunol., 29, 2367, 1999) have observedthat the activation of T cells by an alloantigen in the presence ofantibodies which block the CD80/CD28 or CD86/CD28 and CD40/CD40Linteractions induced an antigen specific anergy which is accompanied bya reduced production of IFN-γ and an increased production of IL-10.

[0012] Chabot et al. (J. Immunol., 162, 6819, 1999) report that theproduction of IL-10 which is induced by the interaction of microglialcells with T lymphocytes is reduced by a blockage of CD40/CD40L,B7/CTLA-4 or B7/CD28, or of CD23.

[0013] The inventors have now developed a novel process which allows toobtain antigen-specific Tr1 lymphocytes, rapidly in an amount which iswell above the amount obtained by the activation of CD4⁺ T cells in thepresence of IL-10. This is because they have found that growing CD4⁺ Tcells in the presence of an inductor antigen, and cells which presentthe antigen expressing a class II HLA molecule and the human LFA-3(CD58) molecule, but which do not express any of the costimulatorymolecules B7-1 (CD80), B7-2 (CD86), B7-H1, CD40, CD23 and ICAM-1 (CD54),induced the differentiation of Tr1 cells which are specific for saidantigen.

[0014] The LFA-3 molecule is the ligand of the CD2 receptor of Tlymphocytes. Two forms of LFA-3 have been described: a transmembraneform (Walner et al., J. Exp. Med. 166, 923-932, 1987; PCT Application WO88/09826), and a form referred to as “PI-linked LFA3”, which is anchoredto the cell membrane by means of a glycolipid containingphosphatidylinositol (PCT Application WO 90/02181).

[0015] The present invention relates to the use of artificialantigen-presenting cells which express a class II molecule of the HLAsystem and a human LFA-3 molecule and which do not express any of thecostimulatory molecules B7-1, B7-2, B7-H1, CD40, CD23 or ICAM-1 (CD54),for obtaining antigen-specific regulatory Tr1 lymphocytes.

[0016] Cells which are defined as antigen-specific regulatory. Tr1lymphocytes are cells which have the following characteristics afterrestimulation with said antigen:

[0017] they produce a large amount of IL-10, viz. an amount of 3×10³pg/10⁶ cells or above, in general 5×10³ to 20×10³ pg of IL-10 per 10⁶cells;

[0018] they produce an amount of IL-2 of 50 pg/10⁶ cells or less; theIL-10/IL-2 ratio produced by said cells being at least 50/1, generally100/1 to 500/1;

[0019] they produce an amount of IL-4 of 50 pg/10⁶ cells or less; theIL-10/IL-4 ratio produced by said cells being at least 300/1, generally500/1 to 10000/1;

[0020] the proliferation of CD4⁺ T cells stimulated by an antigen in thepresence of Tr1 cells is reduced at least 2-fold, in general at least 2to 100-fold.

[0021] In particular, the present invention relates to a method for thepreparation of antigen-specific regulatory Tr1 lymphocytes from thelymphocytes of a patient, characterized in that it comprises:

[0022] the in vitro activation of said lymphocytes in the presence ofthe selected antigen, which is presented by artificialantigen-presenting cells as defined hereinabove, and

[0023] recovering from said lymphocytes a population of activated CD4⁺ Tlymphocytes comprising at least 10%, preferably at least 50%, veryespecially preferably at least 80% of Tr1 lymphocytes which are specificfor said antigen.

[0024] Artificial antigen-presenting cells which can be used forcarrying out the present invention can advantageously be obtained bycotransfecting animal cells which do not express any of the above-statedcostimulation molecules with a nucleic acid sequence encoding the αchain of a class II HLA molecule, a nucleic acid sequence encoding the βchain of a class II HLA molecule and a nucleic acid sequence encodingany of the two forms of LFA-3. If desired, these cells can likewise betransfected with a nucleic acid sequence encoding the antigen againstwhich the specificity of the Tr1 lymphocytes is to be induced. Thesenucleic acid sequences can be borne by different nucleic acid moleculesor else two or more of them can be borne by the same nucleic acidmolecule.

[0025] Animal cells which can be used for obtaining said artificialantigen-presenting cells can be cells of human, autologous orheterologous origin or else cells of xenogeneic origin, in particularmammalian cells. Recently isolated primary cultures can be used; ingeneral, it will be preferred to use established cell lines, which aremore homogeneous, and capable of proliferating over several generations.

[0026] They can take the form of, for example, fibroblasts,keratinocytes, kidney tubule cells, Schwann cells, myoblasts,endothelial cells and the like.

[0027] The class II CMH molecule which is expressed by the artificialantigen-presenting cells will be selected from among the different humanHLA-2 molecules which are available, depending on the HLA class II typeof the patient from which CD4⁺ T lymphocytes are taken and on theintended use of the Tr1 cells.

[0028] In general, artificial antigen-presenting cells will be usedwhich express an HLA-2 molecule which is also expressed by the patient;however, it is also possible to chose an HLA-2 molecule which differsfrom those expressed by said patient; for example, if it is desired toproduce Tr1 cells which can be used for preventing graft rejection, thismay take the form of an HLA-2 molecule expressed by the graft cells; inthis case, the HLA-2 molecule constitutes the antigen against which theTr1 cells obtained will be directed.

[0029] The antigen used for carrying out the method according to theinvention will be chosen depending on the use for which theantigen-specific Tr1 cells to be produced are intended. The antigen cantake the form of an antigen associated with an inflammatory orautoimmune pathology; likewise, it may take the form of an antigenwithout relation to a particular pathology but which can be administeredif the Tr1 cells are to be activated to control an undesirable immuneresponse.

[0030] The artificial antigen-presenting cells can be charged with theantigen in the traditional manner by coincubating the cells and saidantigen. The latter can be present in native form and be prepared bysaid antigen-presenting cells; it can likewise be present in the form ofone or more antigenic peptides which can be attached directly by theHLA-2 molecules which are expressed by said cells. Alternatively, theantigen can be expressed by the artificial antigen-presenting cells whenthe latter have previously been transfected with a nucleic acid sequenceencoding it.

[0031] For carrying out the method according to the invention, the invitro activation of the lymphocytes can be carried out directly on PBMCs(peripheral blood mononuclear cells) taken from said patient. It canalso be carried out on CD4⁺ T lymphocytes which have previously beenisolated from these PBMCs.

[0032] The lymphocytes are activated by coculturing them over 2 to 10days, preferably over 6 to 8 days, in the presence of theabove-described artificial antigen-presenting cells which are chargedwith the selected antigen.

[0033] At the end of this culture period, the activated CD4⁺ Tlymphocytes are selected on the basis of the expression of the CD4³⁰marker and one or more activation markers such as CD25, CD69, CD45RO andthe like.

[0034] At the end of this culture period, a cell population is obtainedin which the Tr1 lymphocytes account for at least 10%, in general forbetween 50 and 80%, of the activated antigen-specific T lymphocytes.

[0035] To concentrate the Tr1 lymphocytes even more in this population,the stimulation under the above-defined conditions may be repeated.

[0036] Thus, a population of activated CD4⁺ T lymphocytes is obtainedwhich comprises at least 30%, in general between 60 and 90%, of Tr1lymphocytes among the antigen-specific T cells.

[0037] Likewise, Tr1 lymphocyte clones can be isolated from thispopulation. These clones can be identified readily on the basis of thecharacteristic cytokine production profile of the Tr1 lymphocytes asdefined hereinabove.

[0038] Clone propagation may be carried out in a nutrient mediumcomprising unspecific growth factors such as IL-4 and IL-2. A preferredembodiment consists in using anti-CD3- and anti-CD28-antibody-coupledbeads as stimulation agent.

[0039] The Tr1 cells obtained by the method according to the inventionpresent all the characteristics of the Tr1 cells obtained by activationof CD4⁺ T lymphocytes in the presence of IL-10 and have therefore thesame applications as the latter for regulating the immune response.

[0040] The present invention also relates to a process for thepreparation of a pharmaceutical composition intended for the treatmentof inflammatory diseases and/or autoimmune diseases, characterized inthat it comprises preparing antigen-specific Tr1 lymphocytes by a methodaccording to the invention and packaging said Tr1 lymphocytes in aformulation which is suitable for their administration to a patient.

[0041] The present invention will be understood better with the aid ofthe remainder of the description which follows, which relates tononlimiting examples which illustrate how the method according to theinvention is carried out.

EXAMPLE 1 Preparation of Tr Lymphocytes

[0042] Isolation of CD4⁺ T Cells

[0043] Peripheral mononuclear cells (PBMCs) are obtained bycentrifugation on Ficoll-Hypaque.

[0044] The CD4⁺ T cells are obtained by eliminating the non-CD4⁺ cellsusing anti-CD8 (L533), anti-CD11b (OKM1), and anti-CD20 (2H7) antibodiesas described in the following protocol. The cells are incubated for 20minutes at 4° C. with saturating antibody concentrations. After washing,Dynabeads (Dynal, Oslo, Norway) are added at a bead/target cell ratio of1/1, and the mixture is incubated for 1 hour at 4° C. The beads whichhave the non-CD4⁺ cells attached to them are eliminated by applicationof a magnetic field. Analysis of the remaining cells by flowcytofluorometry (FACSstar, Becton Dickinson) demonstrates that theycomprise 90 to 95% CD4⁺ T cells.

[0045] Obtaining the Artificial Antigen-Presenting Cells

[0046] Murine L fibroblast cells (ATCC CCL-1) were cotransfected withnucleic acid sequences encoding LFA-3 and a class II HLA molecule.

[0047] Obtaining a LFA-3-Encoding cDNA

[0048] An LFA-3-encoding cDNA was prepared from a complete cDNA libraryof human peripheral mononuclear cells by means of polymerase chainreaction (PCR) using the primer referred to as 5′LFA-3, which comprisesthe sequence of nucleotides 13 to 33 of the sequence encoding humanLFA-3 (Genbank Access Number X06296), flanked 5′ by a KpnI site, and theprimer referred to as 3′LFA-3, which comprises the sequence ofnucleotides 708 to 728 of the sequence encoding human LFA-3, flanked 5′by an NotI site.

[0049] Obtaining a DR1-Encoding cDNA

[0050] A cDNA encoding the DR1 alpha chain was obtained from a completecDNA library of human peripheral mononuclear cells from a DR1⁺ donor bypolymerase chain reaction (PCR) using the primers referred to as DR1A5′and DR1A3′, which comprise the sequence of nucleotides 151 to 176 andthe sequence of nucleotides 769 to 792, respectively, of the sequenceencoding the DR1 alpha chain (Genbank Access Number K01171). A cDNAencoding the DR1 beta chain was obtained in the same manner using theprimers referred to above as DR1B5′ and DR1B3′, which comprise thesequence of nucleotides 52 to 74 and 850 to 923, respectively, of thesequence encoding the DR1 beta chain (Genbank Access Number NM002124).

[0051] Transfection of the Cells

[0052] Each of the cDNAs obtained was cloned into the TA site of avector pcDNA 3.1/hygro (Invitrogen). The vectors obtained were used forcotransfecting the L cells by electroporation.

[0053] The stable transfectants are separated by flow cytofluorometry(FACSVantage SE, Becton Dickinson) by labeling the cells with the aid ofan anti-LFA-3 antibody (1C3) and anti-DR antibody (L243). The cellswhich express LFA-3 and DR simultaneously are retained and cultured onF12 medium (Life Technologies) supplemented with 10% fetal calf serum(Boehringer) with added penicillin and streptomycin.

[0054] Antigen-presenting cells which coexpress LFA-3 and DR1 werelikewise prepared as described above by cotransfecting P815 cells(ATCC-TIB64).

[0055] Untransfected L cells or P815 cells, or cells obtained bytransfection of L cells or P815 cells which express only the DR1molecule, were likewise used by way of control.

[0056] Obtaining the Tr Lymphocytes

[0057] CD4⁺ T cells isolated as described hereinabove from PBMCs of anon-DR1 donor are suspended at a concentration of 2×10⁶ cells/ml inYssel medium (Yssel et al., J. Immunol. Methods, 72, 219-227, 1984). Thecell suspension is divided in a 24-well culture plate at 1 ml per well.The transfected L-DR1-LFA3 cells or the transfected L-DR1 cells, whichhave been obtained as described hereinabove, or, by way of control,B-EBV DR1⁺ lymphoblastoid cells are irradiated (60 GY) and added to eachwell at a concentration of 5×10⁵ cells/ml.

[0058] After incubation for 7 days, the cells are harvested and washedin PBS and then labeled with the aid of anti-CD4 antibody (RPA-T4) andanti-CD25 antibody (M-A251). The CD4⁺CD25⁺ cells are separated by flowcytofluorometry and cloned at 1 cell/well in a 96-well plate in Ysselmedium. Clonal propagation is carried out in the presence of IL-2 andIL-4 by the technique described by Spits and Yssel (J. Immunol. Methods,9, 416-421, 1996). After the propagation of the clones, the differentclones are restimulated with B-EBV DR1⁺ lymphoblastoid cells. 48 hoursafter the stimulation, the cell supernatant is recovered and the profileof the production of cytokines IL-10 and IFN-γ in response to thisstimulation was determined by quantitatively determining these cytokinesby ELISA in accordance with the protocol described by Abrams et al.(Curr. Protocols Immunol., 13, pp 6.1-6-15, 1995).

[0059] The results illustrated in Table I hereinbelow represent theproduction of cytokines IL-10 and IFN-γ by Tr1 cells (mean i standarddeviation of 10 clones) from 2 different donors TABLE I Donor 1 Donor 2IL-10 IFN-γ IL-10 IFN-γ Cell (pg/ml) (pg/ml) (pg/ml) (pg/ml) L-DR1 326 ±40  13372 ± 1500 3657 ± 324 9638 ± 63  L-DR1- 2315 ± 183 19403 ± 9756402 ± 272  914 ± 322 LFA-3 B-EBV <40 16005 ± 123 174 ± 59 4930 ± 685DR1⁺

[0060] In another experimentation series, the CD4⁺ T cells isolated fromPBMCs from a DR1⁺ donor are mixed as described hereinabove withantigen-presenting cells expressing the DR1 molecule and the LFA-3molecule.

[0061] An inductor antigen (peptide HA 307-319, which corresponds to afragment of the capsid antigen of the virus Haemophilus influenzae) isadded to the cell mixture at a concentration of 50 μg/ml.

[0062] After incubation for 3 days, the CD4⁺CD25⁺ cells are separated byflow cytofluorometry and cloned as described hereinabove.

[0063] After the propagation of clones, the different clones arerestimulated with B-EBV lymphoblastoid cells charged with the HA peptide(10 μM). 48 hours after the stimulation, the cell supernatant isrecovered and the profile of the production of the cytokines IL-2, IL-4,IL-10 and IFY-γ in response to this stimulation was determined byquantitative determination of these cytokines by means of ELISA inaccordance with the protocol described by Abrams and co-workers.

[0064] Most (approximately 70%) of the CD4⁺CD25⁺ clones isolated presentthe cytokine production profile of TR1 cells.

[0065] The cytokine production profiles of 9 of these Tr1 clones areillustrated by Table II hereinbelow. TABLE II IL-2 IL-4 IL-10 IFN-γClone (pg/ml) (pg/ml) (pg/ml) (pg/ml) HA-1A12 <20 <40 12358 521 HA-IB6<20 <40 11897 497 HA-IC9 <20 <40 14598 1369 HA-IE5 <20 <40 13549 314HA-IE7 40 <40 11697 876 HA-IF2 <20 <40 10597 1057 HA-2B6 <20 <40 17891697 HA-2D5 32 <40 16589 503 HA-2F2 <20 <40 17803 873

[0066] Tr1 cell clones with the same cytokine production profile havealso been obtained using P815-DR1-LFA-3 cells as antigen-presentingcells, which demonstrates that obtaining Tr1 cells is not linked to thecharacteristics of the cell line from which the antigen-presenting cellsare obtained.

EXAMPLE 2 Immunoregulatory Characteristics of TR1 Lymphocytes Obtainedby the Method According to the Invention

[0067] The immunoregulatory characteristics of antigen-specific Tr1cells were tested as follows:

[0068] Human CD4⁺ T cells from a DR1⁺ donor (1×10⁶ /ml) are stimulatedby irradiated (60 GY) syngenic monocytes (1×10⁶/ml) in the presence ofthe peptide HA 307-319 (50 μM) and of tetanus anatoxin (TT: 50 μg/ml).

[0069] The same experiment is carried out in parallel, adding anHA-specific Tr1 clone obtained as described above to the cells(2×10⁵/ml), alone or in the presence of an antibody directed against theIL-10 receptor (anti-IL-10R, 10 μg/ml), of an anti-TGF-β antibody (20μg/ml) or of a mixture of these 2 antibodies.

[0070] The proliferation of the CD4⁺ T cells is determined after 5 daysby measuring the incorporation of tritiated thymidine.

[0071] The results obtained for 3 HA-specific Tr1 clones are,illustrated in FIG. 1.

[0072] Key to FIG. 1:

[0073] X-axis: proliferation (incorporated tritiated thymidine, in cpm).

[0074] Y-axis:

[0075] Medium: non-stimulated CD4⁺ T cells;

[0076] TT⁺ HA peptide: CD4⁺ T cells stimulated by the peptide HA 307-319and the tetanus anatoxin;

[0077]⁺Tr1 HA specific: CD4⁺ T cells stimulated by the peptide HA307-319 and the tetanus anatoxin in the presence of Tr1 cells which arespecific for the HA antigen;

[0078]⁺anti IL-10R: CD4⁺ T cells stimulated by the peptide HA 307-319and the tetanus anatoxin in the presence of Tr1 cells which are specificfor the HA antigen and of anti-IL-10R antibodies;

[0079]⁺anti-TGF-β: CD4⁺ T cells stimulated by the peptide HA 307-319 andthe tetanus anatoxin in the presence of Tr1 cells which are specific forthe HA antigen and of anti-TGF-β antibodies;

[0080]⁺anti IL-10R⁺anti-TGF-β: CD4⁺ T cells stimulated by the peptide HA307-319 and the tetanus anatoxin in the presence of Tr1 cells which arespecific for the HA antigen and of a mixture of anti-IL-10R antibodiesand anti-TGF-β antibodies.

[0081] These results demonstrate that the Tr1 lymphocytes obtained bythe process according to the invention diminish considerably theantigen-specific proliferation of CD4⁺ T cells. This inhibitory effectis only very weakly attenuated by anti-IR-10R antibodies or anti-TGF-βantibodies alone and is partially annulled by a mixture of the 2antibodies.

1-7. (canceled)
 8. Artificial antigen-presenting cells comprising cellswhich express a class II molecule of the HLA system and a human LFA-3molecule and which do not express any of the costimulatory moleculesB7-1, B7-2, B7-H1, CD40, CD23 or ICAM-1.
 9. Artificialantigen-presenting cells as claimed in claim 8 wherein mammalian cellsthat fail to express B7-1, B7-2, B7-H1, CD40, CD23 or ICAM-1 aretransfected with nucleic acid molecules capable of expressing a class IImolecule of the HLA system and a human LFA-3 molecule.
 10. Artificialantigen-presenting cells as claimed in claim 9 wherein the mammaliancells are selected from the group consisting of fibroblasts,keratinocytes, kidney tubule cells, Schwann cells, myoblasts, andendothelial cells.
 11. Artificial antigen-presenting cells as claimed inclaim 9 wherein the mammalian cells are cotransfected with a nucleicacid sequence encoding the α chain of a class II HLA molecule, a nucleicacid sequence encoding the β chain of a class II HLA molecule and anucleic acid sequence encoding human LFA-3.
 12. Artificialantigen-presenting cells as claimed in claim 8 wherein in addition tothe HLA molecule an additional selected antigen has been associated withthe antigen-presenting cell.
 13. Artificial antigen-presenting cells asclaimed in claim 12 wherein a selected antigen has been associated withthe antigen-presenting cell by a method selected from the groupconsisting of co-incubating the cells and the selected antigen,attaching selected antigenic peptides directly to the expressed HLA-2molecule, and co-transfecting the cells with a nucleic acid moleculeencoding the selected antigen.
 14. A method of preparingantigen-specific regulatory Tr1 lymphocytes by activation anddifferentiation of CD4⁺ lymphocytes of a subject, comprising: separatinglymphocytes from a sample of a subject's blood or serum, activating saidlymphocytes in vitro by co-culturing with the artificialantigen-presenting cells as defined in claim 1, and recovering from saidactivated lymphocytes a population of activated CD4⁺ T lymphocytescomprising at least 10% of Tr1 lymphocytes which are specific for theantigen of the antigen-presenting cells.
 15. The method as claimed inclaim 14, wherein the artificial antigen-presenting cells are obtainedby co-transfection of mammalian cells selected from the group consistingof fibroblasts, keratinocytes, kidney tubule cells, Schwann cells,myoblasts, and endothelial cells, with a nucleic acid sequence encodingthe α chain of a class II HLA molecule, a nucleic acid sequence encodingthe β chain of a class II HLA molecule and a nucleic acid sequenceencoding human LFA-3.
 16. The method as claimed in claim 14, wherein theantigen-presenting cells have a selected antigen associated with themand further comprising activating the lymphocytes in vitro with theselected antigen in addition to the antigen-presenting cells in theactivating step.
 17. The method as claimed in claim 16, furthercomprising repeating the steps in which CD4⁺ T lymphocytes are activatedin vitro with the selected antigen and the antigen-presenting cells, andthe desired Tr1 lymphocyte population are separated, until thepopulation has been enriched to at least 30% Tr1 lymphocytes that arespecific for the selected antigen.
 18. The method as claimed in claim17, further comprising isolating Tr1 lymphocyte clones from therecovered enriched cell population; and propagating the isolated Tr1lymphocyte clones.
 19. A cell population comprising the CD4⁺ Tr1lymphocytes resulting from the process of claim
 14. 20. A cellpopulation comprising the CD4⁺ Tr1 lymphocytes resulting from theprocess of claim
 15. 21. A cell population comprising the CD4⁺ Tr1lymphocytes resulting from the process of claim
 16. 22. A cellpopulation comprising the CD4⁺ Tr1 lymphocytes resulting from theprocess of claim
 17. 23. A cell population comprising the CD4⁺ Tr1lymphocytes resulting from the process of claim
 18. 24. The method oftreating inflammatory diseases and/or autoimmune diseases, comprisingadministering a therapeutically effective amount of the cell populationof claim 19 to a patient in need thereof.
 25. The method of treatinginflammatory diseases and/or autoimmune diseases, comprisingadministering a therapeutically effective amount of the cell populationof claim 20 to a patient in need thereof.
 26. The method of treatinginflammatory diseases and/or autoimmune diseases, comprisingadministering a therapeutically effective amount of the cell populationof claim 21 to a patient in need thereof.
 27. The method of treatinginflammatory diseases and/or autoimmune diseases, comprisingadministering a therapeutically effective amount of the cell populationof claim 22 to a patient in need thereof.
 28. The method of treatinginflammatory diseases and/or autoimmune diseases, comprisingadministering a therapeutically effective amount of the cell populationof claim 23 to a patient in need thereof.